Recently, a large number of measurements using intermolecular interactions such as immune responses are being carried out in clinical tests or the like. Among these, several techniques are preferably used that do not require a complicated operation or a labeled material but are capable of detecting the change in the binding amount of a test substance with high sensitivity. Examples of such techniques include a surface plasmon resonance (SPR) measurement technique, a quartz crystal microbalance (QCM) measurement technique, and a measurement technique using functional surfaces ranging from gold colloid particles to ultra-fine particles. In any of these techniques, the surface on which a substance to be measured is immobilized is important. Hereinafter, the surface plasmon resonance (SPR) measurement technique will be described as an example.
In general, a measurement chip used for measuring a target substance includes a chip in which an evaporated metal film and a thin film having a functional group capable of immobilizing a target substance are formed on a transparent substrate (e.g., glass) in this order. The target substance is immobilized on the surface of the metal film via the functional group. The interaction between substances is analyzed by measuring a specific binding reaction between the target substance and a test substance.
However, the interaction between the target substance and the test substance, which occurs only when a substance assisting the interaction between the target substance and the test substance is present, cannot be directly detected by the method described above. In order to detect such an interaction between the target substance and the test substance, the test substance and an auxiliary substance are required to be simultaneously supplied to the target substance so that the auxiliary substance makes contact with the test substance or the target substance. For this reason, not only is a large amount of the auxiliary substance required, but also the cost of measurement is affected if it is difficult to obtain the auxiliary substance.
In addition, since the auxiliary substance, test substance and target substance are required to be located adjacent to one another, there has also been a problem in that the reaction efficiency decreases.
Moreover, the auxiliary substance includes not only a substance which does not interact with the target substance by itself but also a substance which does interact with the target substance. For this reason, in cases where the auxiliary substance interacts with target substances, there is also a problem in that it is difficult to detect a precise interaction between the target substance and the test substance.
In order to solve these problems, for example, JOURNAL OF MOLECULAR RECOGNITION 1999, Vol. 12, pp. 316-321 discloses a method for preparing and immobilizing an IL-2 receptor complex, in which the extracellular domains of IL-2 receptor subunits are integrated with coiled-coil (leucine zipper) domains. This document also discloses that such a complex exhibits a higher affinity than the subunit alone.
However, in this method there is a problem in that expression level of the desired fusion substance is low because a tag allowing a coiled-coil reaction of the receptor needs to be introduced by a recombination technique. In addition, there is also a problem in that coiled-coil reaction sites inhibit the binding activity of the receptor with a test substance, and the versatility of this method is low.
As a technique employing multiple molecules to capture one test substance, a technique called molecular imprinting is disclosed in Japanese Patent Nos. 3527239 and 4036961, and Japanese Patent Application Laid-Open (JP-A) No. 2006-137805. This molecular imprinting is a method for preparing an artificial molecular recognizing substance, in which a target molecule is immobilized on a substrate using an organic polymer, followed by removing the target molecule, whereby a structure corresponding to the molecular shape of the target molecule is left behind as a porous body.
However, in molecular imprinting, since an antibody is immobilized on a substrate by polymerization, an antigen must penetrate into a cross-linked gel matrix structure in order to reach the antibody. Thus it takes time for the antigen to react with the antibody, thereby requiring a highly concentrated antigen in order for a substance to be recognized for certain. Furthermore, the ability to remove the antigen by washing and responsiveness when the antigen is again applied after removal tend to be decreased.
JP-A No. 10-78436 discloses a method for measuring a concentration of an antigen in which either of a VH-region polypeptide or a VL-region polypeptide is immobilized to a solid phase, which utilizes a phenomenon whereby the stability of the Fv region (a recognition site of the antibody) varies depending on the binding with the antibody.